Lsa蛋白对金黄色葡萄球菌介导的炎性细胞因子表达的影响  

Effects of Lectin Subunit Alpha on the Expression of Staphylococcus aureus-stimulated Inflammatory Cytokines Mediated

作　　者：卫瑞阳 白杰[1,3] 徐彬 席燕燕[1,3] 王琳燚 王改利 付趁[1,3] 魏凤仙 李绍钰 

机构地区：[1]河南省农业科学院畜牧兽医研究所,郑州450002 [2]河南农业大学牧医工程学院,郑州450002 [3]河南省饲料与养殖环境控制工程技术研究中心河南省畜禽繁育与营养调控重点实验室,郑州450002

出　　处：《生物技术通报》2018年第1期215-222,共8页Biotechnology Bulletin

基　　金：国家现代农业产业技术体系项目(CARS-41);国家重点研发计划项目(2016YFD0500509-09);河南省农业科学院优秀青年基金项目(2016YQ20)

摘　　要：试验旨在研究家蝇Lsa(Lectin subunit alpha)蛋白在金黄色葡萄球菌(Staphylococcus aureus)刺激下的炎性反应。通过慢病毒转染的方法,将构建好的载体pLEX-Lsa及pLEX分别转染到RAW264.7细胞中,通过筛选,获得稳定表达的RAWpLEX-Lsa及RAW-pLEX细胞系,其中RAW-pLEX细胞作为阴性对照。然后通过S.aureus刺激细胞,收集未刺激和刺激后3 h,6h,12 h和24 h的细胞,且收集未刺激和刺激6 h细胞上清。提取RNA,通过实时定量PCR(RT-PCR)检测炎性细胞因子TNF-a,IL-1β,NFκB-1和NFκB-2基因的相对转录水平。通过酶联免疫吸附测定(ELISA)检测细胞上清中TNF-a和IL-1β的蛋白水平。RT-PCR结果显示,在S.aureus刺激后,与RAW-pLEX细胞相比,RAW-pLEX-Lsa细胞中TNF-a的两个转录剪接体的相对转录水平,TNF-α-tv-1在6 h显著下调(P<0.05),TNF-α-tv-2在6 h和12 h显著下调(P<0.05);RAW-pLEX-Lsa细胞中IL-1β的两个转录剪接体的相对转录水平,IL-1β-T在3 h,6 h和12 h显著下调(P<0.05),IL-1β在3 h和6 h显著下调(P<0.05);RAW-pLEX-Lsa细胞中NFκB-1和NFκB-2基因的相对转录水平无明显下降趋势。ELISA检测结果显示,在S.aureus刺激6 h后,与RAW-pLEX细胞相比,RAW-pLEX-Lsa细胞上清中,TNF-a的蛋白水平显著下调(P<0.05)。结果表明,家蝇Lsa蛋白可以有效地发挥抗炎活性,显著地抑制TNF-α和IL-1β的转录和生产,而TNF-α和IL-1β的转录和生产的下调并不是通过抑制NFκB信号通路的活性引起的。The attempt of this work is to investigate the anti-inflammatory activity of Musca domestica lectin subunit alpha （ Lsa ） toward Staphylococcus aureus. The well-constructed pLEX-Lsa and pLEX were transfected into RAW264.7 ceils with recombinant lentiviruses, then the stably-expressed RAW-pLEX-Lsa and RAW-pLEX were obtained after screening, and RAW-pLEX was as a negative control. S. aureus was used to stimulate RAW-pLEX-Lsa ceils and RAW-pLEX ceils, then both un-stimulated cells and stimulated cells for 3 h, 6 h, 12 h, and 24 h were collected, and the cell culture supernatants of 6 h un-stimulated and stimulated cells were also collected. Real-time quantitative reversetranscription PCR （ RT-PCR ） was performed to analyze the mRNA expression of TNF-α, IL-1β, NFκB-1 and NFκB-2, and the protein level of TNF-α and IL-1β in the supernatant were detected by ELISA. The results of RT-PCR showed that the stimulation of RAW-pLEX-Lsa by S. aureus significantly down-regulated the mRNA expression of TNF-a transcript variant 1 （ TNF-α-tv-1 ） at 6 h （ P〈0.05 ） and TNF-α transcript variant 2 （ TNF-α-tv-2 ） at 6 h and 12 h （ P 〈 0.05 ）, compared with the RAW-pLEX cells. The stable transfection of Lsa in RAW264.7 cells stimulated by S. aureus significantly down-regulated the mRNA expression of IL-1β-T at 3 h, 6 h and 12 h （ P 〈 0.05 ） and the mRNA expression of 1L-1β at 3 h and 6 h （ P 〈 0.05 ）, compared with the RAW-pLEX cells, while the mRNA expression of NFκB-1 and NFκB- 2 in RAW-pLEX-Lsa had insignificant decreasing. The results of ELISA denmnstrated that the protein level of TNF-α in the supernatants of PtAW-pLEX-Lsa cells at 6 h after stimulation by S. aureus significantly down-regulated （ P 〈 0.05 ） . In conclusion, Lsa possesses potenl anti- inflammatory activity, and stable transfection of Lsa in RAW264.7 cells inhibits the expression and production of TNF-a and IL-lfl. However, the inhibition of TNF-α and IL-lfl is not resulted from blocking the activation of NF

关 键 词：凝集素 RAW264.7 炎性细胞因子 NFΚB 转录剪接体 

分 类 号：S852.61[农业科学—基础兽医学]